Molecular cloning of the tomato Hairless gene implicates actin dynamics in trichome-mediated defense and mechanical properties of stem tissue

Journal of Experimental Botany, Vol. 67, No. 18 pp. 5313–5324, 2016 doi:10.1093/jxb/erw292 Advance Access publication 31 July 2016 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER Molecular cloning of the tomato Hairless gene implicates actin dynamics in trichome-mediated defense and mechanical properties of stem tissue Jin-Ho Kang1,2,*, Marcelo L. Campos1,‡, Starla Zemelis-Durfee1,3, Jameel M. Al-Haddad3, A. Daniel Jones4,5, Frank W. Telewski3, Federica Brandizzi1,3 and Gregg A. Howe1,5,* 1 Department of Energy-Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA Graduate School of International Agricultural Technology and Crop Biotechnology Institute/GreenBio Science and Technology, Seoul National University, Pyeongchang 25354, Republic of Korea 3 Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA 4 Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA 5 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA 2 Received 24 May 2016; Accepted 9 July 2016 Editor: Christine Raines, University of Essex Abstract Trichomes are epidermal structures that provide a first line of defense against arthropod herbivores. The recessive hairless (hl) mutation in tomato (Solanum lycopersicum L.) causes severe distortion of trichomes on all aerial tissues, impairs the accumulation of sesquiterpene and polyphenolic compounds in glandular trichomes, and compromises resistance to the specialist herbivore Manduca sexta. Here, we demonstrate that the tomato Hl gene encodes a subunit (SRA1) of the highly conserved WAVE regulatory complex that controls nucleation of actin filaments in a wide range of eukaryotic cells. The tomato SRA1 gene spans a 42-kb region containing both Solyc11g013280 and Solyc11g013290. The hl mutation corresponds to a complex 3-kb deletion that removes the last exon of the gene. Expression of a wild-type SRA1 cDNA in the hl mutant background restored normal trichome development, accumulation of glandular trichomederived metabolites, and resistance to insect herbivory. These findings establish a role for SRA1 in the development of tomato trichomes and also implicate the actin-cytoskeleton network in cytosolic control of specialized metabolism for plant defense. We also show that the brittleness of hl mutant stems is associated with altered mechanical and cell morphological properties of stem tissue, and demonstrate that this defect is directly linked to the mutation in SRA1. Key words: Actin-cytoskeleton network, cell wall, mechanical properties, plant-insect interaction, specialized metabolism, terpenoid, tomato (Solanum lycopersicum), trichome. Introduction Trichomes are epidermal outgrowths that exhibit remarkable morphological variation between plant species. In addition to being either unicellular or multicellular, trichomes are often distinguished as being nonglandular (simple hairs) or glandular, with the latter involved in the production and secretion of a wide spectrum of specialized metabolites. Trichomes have © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: , Present address: Departamento de Botânica, Instituto de Ciências Biológicas, Universidade de Brasília, 70910-900, Brasília, Distrito Federal, Brazil ‡ 5314 | Kang et al. trichome density (Paetzold et al., 2010). The tomato Woolly gene encodes a homeodomain-containing transcription factor that specifically controls the development of type I trichomes (Yang et al., 2011). These collective findings indicate that the development of various trichome types in tomato involves regulatory processes that are specific to a particular structure. A better understanding of trichome development and function could be achieved through the study of genes that affect multiple trichome types in a single plant species. The hairless (hl) mutant of tomato was identified more than a half-century ago as a spontaneous mutant that is defective in trichome production on stems and hypocotyls (Rick and Butler, 1956). Reeves (1977) noted that the ‘hairless’ phenotype of the mutant is not caused by the absence of trichomes but rather the development of trichomes that are severely bent and shortened, which imparts a granular appearance to epidermal surfaces (Fig. 1). Subsequent studies showed that hl plants are deficient in the accumulation of sesquiterpene and polyphenolic compounds within type VI glandular trichomes and that this metabolic phenotype is associated with decreased resistance to insect herbivory (Kang et al., 2010b). Here, we used a map-based cloning approach to demonstrate that Hl encodes the highly conserved SRA1 (specifically Rac1associated protein) subunit of the WAVE regulatory complex (WRC), which is known to control branching of actin filaments in response to various signal inputs at the cell surface. Transgenic complementation experiments showed that SRA1 Fig. 1. Light micrographs of trichomes on the leaf, stem, and hypocotyl of WT and hl plants. Photographs show the adaxial leaf surface (upper row), stem (middle row), and hypocotyl (lower row) of each genotype. Arrows indicate representative type I trichomes. Scale bar, 2 mm. Three-week-old plants were used for all photographs. (This figure is available in color at JXB online.) been implicated in several physiological functions related to biotic and abiotic stress resilience. For example, trichomes protect plants against a variety of arthropod herbivores that depend on plants as a source of nutrition, prevent water loss and may also provide protection from UV-B radiation (Karabourniotis et al., 1992; Werker, 2000; Kennedy, 2003). Molecular genetic studies performed in Arabidopsis have provided detailed insight into the regulatory pathways that control the development of simple, unicellular trichomes. These studies have identified multi-protein transcriptional complexes that control the differentiation of trichomes from epidermal pavement cells (Hulskamp et al., 1994; Marks, 1997; Ramsay and Glover, 2005; Yang and Ye, 2013). Characterization of trichome distortion mutants has further revealed a role for the actin-cytoskeleton network in trichome branching, cell expansion, and other aspects of cell morphogenesis (Mathur and Chua, 2000; Mathur et al., 2003; Szymanski, 2005). Increasing interest in multicellular glandular trichomes as ‘biofactories’ for the production of specialized metabolites has stimulated efforts to understand developmental and metabolic processes tha (...truncated)